The relationship of mutations in the MTHFR, prothrombin, and PAI-1 genes to plasma levels of homocysteine, prothrombin, and PAI-1 in children and adults.

Studies in adults have demonstrated that the genetic mutations C677T methylenetetrahydrofolate reductase (MTHFR), prothrombin 20210A, and the 4G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene are associated with elevated plasma levels of homocysteine. prothrombin and PAI-1, respectively and with an increased risk of thrombosis. No similar data is available in children. Therefore, we assessed the relationship of plasma levels of homocysteine, prothrombin and PAI-1 with their respective mutations in 197 normal children, compared to 40 adults. By stepwise multiple regression, homocysteine was positively associated with age, PAI-1 activity was negatively associated with age, while PAI-1 antigen and prothrombin levels were associated with gender, being higher in girls than boys. When the genotypes were added to the regression model as additional explanatory variables, the MTHFR genotype accounted for 2.9% of the variance of homocysteine (p = 0.024), and the PAI-1 gene accounted for 2.7% of the variance of PAI-1 antigen levels (p = 0.023). Of children homozygous for the MTHFR mutation, 35% had homocysteine levels > or = the age-specific 95th percentile, compared to 2% heterozygotes and 5% wild type normals (p = 0.0001). The mean homocysteine level was higher in children homozygous for the MTHFR gene (8.4 micromol/1) than in heterozygotes (5.5 micromol/l), p <0.05. Of children homozygous for the 4G polymorphism of the PAI-1 gene, 19% had PAI-1 activity levels > or = the age-specific 95th percentile, compared to 2% of heterozygotes and 3% of wild type normals (p = 0.003). Studies of the incidence of the MTHFR, prothrombin, and PAI-1 4G/5G genotypes in children with thrombosis, when compared to these healthy normals, will provide evidence as to which of these genes are associated with thrombophilia.

Sensitivity, specificity and predictive value of modified assays for activated protein C resistance in children.

Very little data is available assessing the clinical utility of coagulation-based APC resistance assays compared to DNA-based analysis for the factor V Leiden mutation in children. Therefore, the clinical utility of four aPTT-based assays for APC resistance was evaluated in 169 children, ages 3 months through 16 years. The prevalence of the Arg506 to Gln mutation was 7/169 (4.1%). Using cutoff points derived from the normal PCR-screened population (n = 162), two assays for APC resistance (APC-SR and n-APC-SR) gave poor concordance with the PCR assay (sensitivity 29% and 57%, respectively). Two modified assays (FDAPC-SR and n-FDAPC-SR), in which patient plasma was prediluted 1:5 in factor V deficient plasma, gave excellent concordance (sensitivity 100%). The predictive value of a positive test was 0.25, 0.44, 1.00 and 0.88 for the APC-SR, n-APC-SR, FDAPC-SR and n-FDAPC-SR, respectively. The FDAPC-SR and n-FDAPC-SR tests gave excellent discrimination using cutoff values derived from the total population (n = 169) without regard to previous PCR screening results.

Factors affecting blood stem cell collections following high-dose cyclophosphamide mobilization in lymphoma, myeloma and solid tumors.

Sixty patients with malignancy were enrolled in a study of high-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT). Stem cells were harvested prior to PBSCT using high-dose cyclophosphamide (CY) mobilization (4 or 7 g/m2) with collection of a median of 4.6 x 10(8)/kg mononuclear cells (range 0.2-9.5) and 21.6 x 10(4)/kg colony forming unit-granulocyte/macrophage (CFU-GM) (range 0.1-220). Forty-seven patients were mobilized once, 11 required two cycles and two required three cycles. Eight patients (13%) failed to reach the optimum CFU-GM target (greater than 15 x 10(4)/kg) following CY mobilization. A number of factors identified those patients who were likely to achieve optimum CFU-GM collections with CY mobilization. These included the use of the higher CY mobilization dose, a longer interval from last chemotherapy cycle to mobilization, and a higher premobilization bone marrow CFU-GM level. Patient's age, the degree of bone marrow infiltration, the nature of disease or the number of pre-mobilization chemotherapeutic cycles did not affect the ability to collect optimum CFU-GM numbers. Whilst the mobilization procedure was associated with moderate non-hematologic toxicity, significant hematological morbidity was observed primarily in patients mobilized using the 7 g/m2 dose. Refinements to the protocol, in particular the use of hematopoietic growth factors, are currently under investigation.